1
Supplementary on line information
Chemicals
Unless otherwise indicated, all chemicals were purchased from Sigma (Sigma-Aldrich,
Milan, Italy).
Oocyte Recovery and In Vitro Oocyte Maturation (IVM)
Cumulus oocyte complexes (COCs) from ovaries of slaughtered mares were recovered
by scraping the follicle wall with a Jacobson curette. Following washing in TCM199
supplemented with 25 mM hepes, 1 mg/ml BSA and 10 µg/ml heparin the COCs were
matured for 22-24 h in TCM199 supplemented with 10% fetal calf serum (FCS), ITS
(insulin, transferrin, sodium selenite), 1mM sodium pyruvate, 100 ng/ml Long-IGF1, 50
ng/ml Long-EGF, and 0.1 IU/ml each of FSH and LH (Pergovet, Serono, Italy), in 4well plates at 38.5°C in an atmosphere of 5% CO2 in air.
The matured oocytes were partially denuded from cumulus cells by pipetting in hepesbuffered SOF (H-SOF)1, supplemented with 25µg/ml hyaluronidase, then exposed to
2.5mg/ml trypsin in PBS for 2 min and the final denudation was performed in H-SOF
with 10% FCS with a fine pipette. Oocytes with an extruded first polar body were
selected for nuclear transfer.
Enucleation
Oocytes were stained with Hoechst 33342 (5 µg/ml) in the presence of cytochalasin B
(5 µg/ml) for 5 minutes then moved in medium containing only cytocalasin B to
complete enucleation within 30 min. Enucleation was performed by the aspiration of
2
metaphase II plate, visualised under UV light, in a minimal volume of cytoplasm by a
micropipette of 20 to 25µm in diameter. All manipulations were in H-SOF.
Cell culture and preparation of donor cells
Adult fibroblast cells were isolated from skin samples and cultured in DMEM/TCM199
1:1, supplemented with 10% FBS for 1-10 passage at 38,5oC in an atmosphere of in 5%
CO2 and 5% O2 in air. The pregnancy that went to term was from passage 4, the one that
aborted was from passage 9. Before nuclear transfer cells were serum starved for 1-4
days by reducing the concentration of the fetal calf serum to 0.5% in the culture
medium. A single cell suspension was prepared by standard trypsinisation at the time of
use.
Nuclear transfer, electrofusion and oocyte activation
Zona pellucida of matured enucleated oocytes was removed by 0,5% pronase. At 25-27
h of IVM fibroblast-cytoplast (enucleated oocyte) pairs were made using 200 µg/ml
phytohemagglutinin P2. The fibroblast-cytoplast pairs were washed in 0.3 M mannitol
solution ( containing 50 µM Ca++ and 100 µM Mg++) and fused by double DC-pulse of
1,3 Kv/cm applied for 30 µsec in a fusion chamber with two platinum electrode 280µm
apart. After pulsing, the pairs were transferred into H-SOF waiting fusion to complete.
Usually 70% fused, the non-fused were subjected to a second round of fusion 20-30
minutes after the first. Overall fusion rate was 97%. At 28-29 h of maturation
reconstructed embryos were activated with 5 µM ionomycin for 4 min followed by 4 h
culture in 1 mM 6-DMAP and 5µg/ml of Cycloheximide in m-SOFaa1.
3
Embryo culture.
Cloned embryos were cultured in 3 µl microdrops of m-SOFaa1 in 5% CO2 and 5% O2
in humidified air at 38.5°C under mineral oil. During embryo culture half of the
medium was renewed on day 3 with fresh m-SOFaa and on day 6 with
DMEM/TCM199 (1:1) supplemented with 5% FCS (day 0 was the day of activation).
Cleavage was assessed at 48 h and the blastocyst rate was recorded on day 8 (table 1).
Embryo transfer
Recipient mares were checked daily by ultrasonography for natural ovulation. Five to
six days after ovulation one or two blastocysts were transferred non-surgically to the
uterine horn ipsilateral to the corpus luteum of each recipient. On day 21 the mares were
scanned (5 MHz linear probe, Sonovet 600, Medison) for pregnancy diagnosis, again on
day 30 and at monthly intervals thereafter.
Monitoring of the pregnancy and delivery
At 315 day of gestation the pregnancy was monitored by ultrasonography with a 3Mhz
convex probe, (Aloka SSD 500). Placenta thickness , size of orbital ring and aorta, heart
beat and presentation of the fetus were checked. All the parameters fell within the
expected range. Delivery started spontaneously at h 1.30 am of the 28th of May 2003
and was completed very quickly without the need of assistance; the foal was standing in
30 minutes and was suckling the mother one hour after birth.
Microsatellite analysis
4
DNA was extracted, using standard protocols, from the blood of the mare, of the foal
and from placenta. Analysis of microsatellite loci was performed using the
StockMarksTM Kit for Horse (Perkin-Elmer, Applied Biosystems) which is based on 12
microsatellite (table 1). The products of DNA amplification by polymerase chain
reaction (PCR) were analysed using an ABIPRISM 377 Applied Biosystems sequencer
with an internal standard GenScan ROX 350bp. The results were analysed by
Genotyper software (Perkin-Elmer Corporation).
1. Gardner DK, Lane M, Spitzer A, Batt PA. Enhanced rates of cleavage and
development for sheep zygotes cultured to the blastocyst stage in vitro in the
absence of serum and somatic cells: amino acids, vitamins, and culturing
embryos in groups stimulate development. Biol Reprod. 1994 Feb;50(2):390400.
2. Booth PJ, Tan SJ, Reipurth R,Holm P, Callesen H. Simplification of bovine
somatic cell nuclear transfer by application of a zona-free manipulation
technique. Cloning Stem Cells 2001, 3: 139-150.
Table 1: Analysis of horse genetic markers
mare
cloned foal
(blood)
(blood)
placenta
Allele 1
Allele 2
Allele1
Allele2
Allele 1
Allele 2
VHL20
90
103
90
103
90
103
HTG4
130
130
130
130
130
130
AHT4
152
154
152
154
152
154
5
HMS7
179
179
179
179
179
179
HTG6
100
100
100
100
100
100
HMS6
161
163
161
163
161
163
HTG7
120
128
120
128
120
128
HMS3
166
166
166
166
166
166
AHT5
131
134
131
134
131
134
ASB2
238
246
238
246
238
246
HTG10
110
110
110
110
110
110
HMS2
225
225
225
225
225
225
Analysis of microsatellite loci was performed using the StockMarksTM Kit for Horse
(Perkin-Elmer, Applied Biosystems). The PCR products were analysed by Genotyper
software (Perkin-Elmer Corporation). The size of each allele is expressed in base pairs.